1. Field of the Invention
The present invention relates to a method of isomerizing glucose with a microbial cell having glucose isomerase immobilized therein.
As already known, glucose isomerase is produced by certain kinds of bacteria, such as Actinomycetes, and accumulated within their cells. Therefore, it is possible to use cells obtained by cultivation as a crude enzyme source for isomerizing glucose. In usual microbial cells, however, the amount of enzyme which flows out of cells during a batch reaction and impossible to be recovered is substantial, though a part of the enzyme remains in the cells, and it can be recovered and re-used. Generally, the liberation of the enzyme from microbial cells becomes extremely large when the cells are used after drying. Subsequently, it is clear that if enzyme liberation from microbial cells were prevented by an immobilization technique and these cells obtained in a dry state, economy and efficiency in enzyme utilization, including transportation, storage and use, would be improved substantially.
2. Description of the Prior Art
By immobilizing an enzyme, its long-term use or repeated use by recovery is made possible and advantages such as reduction of reaction time, continuation and automatization of reaction, etc., are realized. Therefore, various methods for immobilization have been tried with different kinds of enzymes. With regard to glucose isomerase, various methods for immobilization have been reported for cell-free enzymes as well as intracellular enzymes.
Adsorption, entrapment and covalent bonding methods and others have been tried with free glucose isomerase. However, as glucose isomerase is an intracellular enzyme as above-mentioned, it is more convenient if this enzyme can be immobilized within a microbial cell and retained for a long time. Whole cell immobilization does not necessitate a process such as extraction or purification of the enzyme to obtain a cell-free enzyme, and is also free from enzyme inactivation or enzyme leakage at the time of combining the enzyme with a carrier.
As the methods for immobilizing intracellular glucose isomerase, the method of heat-treating microbial cells (Y. Takasaki and A. Kambayashi; Rep. Ferment. Res. Inst. (Japan), 37, 31-37 (1969)), treating microbial cells with a sulfite (N.E. Lloyd, L.T. Lewis, R.M. Logan and D.N. Patel; U.S. Pat. No. 3,694,314, Sept. 26, 1972), incorporating microbial cells into a water-insoluble polymer (Jun Owaki, Yoshiro Minami; Japanese Laid Open Patent Gazette; No. 29,786/1975, Mar. 25, 1975; W.R. Vieth, S.S. Wang and R. Saini; Biotech. Bioeng. 15, 565 (1973)), flocculating microbial cells with a polyelectrolyte (C.K. Lee and M.E. Long; U.S. Pat. No. 3,821,086; June 28, 1974), and treating microbial cells with various metal salts (W.R. Lamm, L.G. Davis and R.G. Dworschak; U.S. Pat. No. 3,821,082; June 28, 1974), etc., are known to date.
The method of the present invention is an extremely simple and effective one, in which intracellular glucose isomerase is immobilized by specific means completely different from conventional methods.